The present invention relates to peptides, which can be used to diagnose, prevent, and treat amyloid-associated diseases, such as Type II diabetes mellitus.
Amyloid material deposition (also referred to as amyloid plaque formation) is a central feature of a variety of unrelated pathological conditions including Alzheimer's disease, prion-related encephalopathies, type II diabetes mellitus, familial amyloidosis and light-chain amyloidosis.
Amyloid material is composed of a dense network of rigid, nonbranching proteinaceous fibrils of indefinite length that are about 80 to 100 Å in diameter. Amyloid fibrils contain a core structure of polypeptide chains arranged in antiparallel β-pleated sheets lying with their long axes perpendicular to the long axis of the fibril [Both et al. (1997) Nature 385:787-93; Glenner (1980) N. Eng. J. Med. 302:1283-92].
Approximately twenty amyloid fibril proteins have been identified in-vivo and correlated with specific diseases. Amyloid proteins share little or no amino acid sequence homology, however the core structure of the amyloid fibrils is essentially the same.
The common core structure of amyloid fibrils and the presence of common substances in amyloid deposits suggest that data characterizing a particular form of amyloid material may also be relevant to other forms of amyloid material and thus can be implemented in template design for the development of drugs against amyloid-associated diseases such as type II diabetes mellitus, Alzheimer's dementia or diseases and prion-related encephalopathies.
Type II diabetes mellitus is a heterogeneous and multifactoral disease characterized by abnormalities in the action of insulin (i.e., insulin resistance) and the secretion of insulin (i.e., beta-cell failure). The relative contribution of each abnormality varies among patients as well as during the course of the disease [Ferrannini (1998) Endocr. Rev. 19:477-90].
Apparently, deposition of islet amyloid is involved in the pathogenesis of type II diabetes. Islet amyloidosis in patients with type II diabetes is associated with a reduced mass of insulin-producing beta cells and is most likely an important factor in the development of beta cell failure. Patients who require insulin treatment have the greatest reduction in islet mass and the most prominent amyloid deposits, indicating that the degree of islet amyloidosis may be related to the severity of the disease [Westermark (1994) Amyloid 1:47-60]. A link between islet amyloidosis and type II diabetes is further supported by the finding of islet amyloid in other animal species in which type II diabetes occurs, notably monkeys and cats [Westermark et al. (1990) Proc. Natl. Acad. Sci. USA 87:5036-40].
The building block of islet amyloid fibrils is a 37 amino acid residue peptide known as islet amyloid polypeptide [IAPP, Johnson et al. (1989) N. Eng. J. Med. 321:513-8]. The nucleotide sequence of the gene indicates that the islet amyloid polypeptide in normal subjects is identical to the islet amyloid polypeptide in amyloid deposits in diabetic patients. This finding suggests that a change in the amino acid sequence of islet amyloid polypeptide is not the pathogenic mechanism that leads to the formation of islet amyloid fibrils [Mosselman et al. (1988) FEBS Lett. 239:227-32].
However, comparison of the amino acid sequences of islet amyloid polypeptide among various animal species, in some of which islet amyloid does not develop, in combination with experiments involving in vitro formation of fibrils from synthetic islet amyloid polypeptide molecules has led to the identification of an “amyloidogenic” region within the human islet amyloid polypeptide molecule that is essential for the formation of fibrils [Johnson et al. (1989) N. Eng. J. Med. 321:513-8; Moriarty et al. (1999) Biochemistry 38:1811-8].
As suggested supra, islet amyloidosis is involved in the loss of up to 50% of beta cell mass in the pancreatic tissue of patients with type II diabetes as well as in diabetic cats and transgenic mice that produce human islet amyloid polypeptide [Hoppener et al. (2000) N. Eng. J. Med. 343:411-19].
Preventing or arresting the process of amyloid-related beta-cell failure at an early stage of type II diabetes might preserve endogenous insulin production and prevent or at least delay hyperglycemia.
Furthermore, it was found that mutations in the IAPP gene are correlated with predisposition and early onset of type II diabetes [Seino (2001) Diabetologia 44:906-9]. While Type II diabetes usually occurs at the age of 50 and higher, individuals with the genetic predisposition may be diagnosed with diabetes at their 30s. Therefore, prevention of amyloid formation may serve as a prophylactic treatment for such individuals (e.g., about 1% of the Far Asian population (China, Korea, Japan, and Taiwan).
Amyloid deposits do not appear to be inert in vivo, but rather are in a dynamic state of turnover and can even regress if the formation of fibrils is halted [Gillmore et al. (1997) Br. J. Haematol. 99:245-56].
Thus, therapies designed to inhibiting the production of human islet amyloid polypeptide or inhibiting amyloidosis may be useful for treating type II diabetes mellitus.
Inhibition of the Production of Islet Amyloid Polypeptide—
Both human islet amyloid gene and insulin share common promoter elements [Mosselman et al. (1988) FEBS Lett. 239:227-32]. Thus, the design of drugs, which inhibit the expression of the islet amyloid polypeptide gene without simultaneously inhibiting the expression of the insulin gene has not been attempted. Nevertheless, direct inhibition of the production of islet amyloid polypeptide may be accomplished through the use of antisense oligonucleotides against human islet amyloid polypeptide messenger RNA (mRNA). In vitro, the addition of antisense oligonucleotides or the expression of antisense complementary DNA against islet amyloid polypeptide mRNA increased the insulin mRNA and protein content of cells, demonstrating the potential effectiveness of this approach [Kulkarni et al. (1996) J. Endocrinol. 151:341-8; Novials et al. (1998) Pancreas 17:182-6]. However, no experimental results demonstrating the in vivo effectiveness of such antisense molecules have been demonstrated.
Inhibition of the Formation of Amyloid Fibrils—
Amyloid, including islet amyloid, contains potential stabilizing or protective substances, such as serum amyloid P component, apolipoprotein E, and perlecan. Blocking their binding to developing amyloid fibrils could inhibit amyloidogenesis [Kahn et al. (1999) Diabetes 48:241-53], as could treatment with antibodies specific for certain parts of an amyloidogenic protein [Solomon et al. (1997) Proc. Natl. Acad. Sci. USA 94:4109-12].
The following summarizes current attempts to engineer drugs having the capability of destabilizing amyloid structures.
Destabilizing Compounds—
Heparin sulfate has been identified as a component of all amyloids and has also been implicated in the earliest stages of inflammation-associated amyloid induction. Kisilevsky and co-workers (Mature Med. 1:143-148, 1995) described the use of low molecular weight anionic sulfonate or sulfate compounds that interfere with the interaction of heparin sulfate with the inflammation-associated amyloid precursor and the β peptide of Alzheimer's disease (AD). Heparin sulfate specifically influences the soluble amyloid precursor (SAA2) to adopt an increased β-sheet structure characteristic of the protein-folding pattern of amyloids. These anionic sulfonate or sulfate compounds were shown to inhibit heparin accelerated Aβ fibril formation and were able to disassemble preformed fibrils in vitro, as monitored by electron micrography. Moreover, these compounds substantially arrested murine splenic inflammation-associated amyloid progression in vivo in acute and chronic models. However, the most potent compound [i.e., poly-(vinylsulfonate)] showed acute toxicity. Similar toxicity has been observed with another compound, IDOX (Anthracycline 4′-iodo-4′-deoxy-doxorubicin), which has been observed to induce amyloid resorption in patients with immunoglobin light chain amyloidosis (AL) [Merlini et al. (1995) Proc. Natl. Acad. Sci. USA].
Destabilizing Antibodies—
Anti-β-amyloid monoclonal antibodies have been shown to be effective in disaggregating β-amyloid plaques and preventing β-amyloid plaque formation in vitro (U.S. Pat. No. 5,688,561). However, no experimental results demonstrating the in vivo effectiveness of such antibodies have been demonstrated.
Destabilizing Peptides—
The finding that the addition of synthetic peptides that disrupt the β-pleated sheets (“β-sheet breakers”) dissociated fibrils and prevented amyloidosis [Soto et al. (1998) Nat. Med. 4:822-6] is particularly promising from a clinical point of view. In brief, a penta-residue peptide inhibited amyloid beta-protein fibrillogenesis, disassembled preformed fibrils in vitro and prevents neuronal death induced by fibrils in cell culture. In addition, the beta-sheet breaker peptide significantly reduced amyloid beta-protein deposition in vivo and completely blocked the formation of amyloid fibrils in a rat brain model of amyloidosis.
While reducing the present invention to practice, the present inventors have demonstrated that contrary to the teachings of U.S. Pat. No. 6,359,112 to Kapurniotu, peptide aggregation into amyloid fibrils is governed by aromatic interactions. Such findings enable to efficiently and accurately design peptides which can be used to diagnose and treat amyloid-associated diseases.